Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfa ces is a key step in the reverse cholesterol transport process, as the subs equent cholesterol esterification reaction drives the removal of cholestero l from tissues into plasma, In this study, the surface plasmon resonance me thod was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apo lipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL ), and small unilamellar phosphatidylcholine vesicles, with biotin tags, we re immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT conce ntration. In addition, three mutants of LCAT (T123I, N228K, and (Delta 53-7 1) were examined in their interactions with LDL. For the wild-type LCAT, bi nding to all lipid surfaces had the same association rate constant, k(a), b ut different dissociation rate constants, k(d), that depended on the presen ce of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, inc reased ionic strength of the buffer decreased K-a for the binding of LCAT t o apoA-I rHDL, For the LCAT mutants, the Delta 53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N 228K) had nearly normal binding to LDL, In conclusion, the association of L CAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT fro m lipoproteins is decreased due to the presence of apoA-I, suggesting prote in-protein interactions. Also, the region of LCAT between residues 53 and 7 1 is essential for interfacial binding.